Despite the extensive hepatic differentiation potential of human umbilical cord lining-derived mesenchymal stem cells (hUC-MSC), little is known about the molecular mechanisms of hUC-MSC differentiation. At the post-transcriptional level, microRNAs are key players in the control of cell fate determination during differentiation. In this study, we aimed to identify microRNAs involved in the hepatic differentiation of hUC-MSCs. After successfully isolating hUC- MSCs, we induced hepatocyte formation in vitro with growth factors. After 26 days of induction, hUC-MSCs could express hepatocyte-specific genes, synthesize urea and glycogen and uptake low-density lipoprotein. Cellular total RNA from hUC-MSCs and hepatic differentiated hUC-MSCs was collected at 7 time points, including 2 days, 6 days, 10 days, 14 days, 22 days and 26 days, for microRNA microarray analysis. Dynamic microRNA profiles were identified that did not overlap or only partially overlapped with microRNAs reported to be involved in human liver development, hepatocyte regeneration or hepatic differentiation of liver-derived progenitor cells. A total of 61 microRNAs among 1205 human and 144 human viral microRNAs displayed consistent changes and were altered at least 2-fold between hUC-MSCs and hepatic differentiated hUC-MSCs. Among these microRNAs, 25 were over-expressed; this over-expression occurred either gradually or increased sharply and was maintained at a high level. A total of 36 microRNAs were under-expressed, with an expression pattern similar to that of the over-expressed microRNAs. The expression of the altered expressed microRNAs was also confirmed by quantitative reverse-transcription polymerase chain reaction. We also found that microRNAs involved in hepatic differentiation were not enriched in hepatocyte or hepatocellular carcinoma cells and can potentially target liver-enriched transcription factors and genes. The elucidation of the microRNA profile during the hepatic differentiation of hUC-MSCs provides the basis for clarifying the role of microRNAs in hUC-MSC hepatic differentiation and specific microRNA selection for the conversion of hUC-MSCs to hepatocytes.The present study was approved by the ethics committee of Xijing Hospital of The Fourth Military Medical University (Xi’an, China). Human umbilical cords were collected after full-term deliveries with informed written consent of the mothers (patient consent in Chinese/English and ethics statement from the ethics committee was shown in Supporting Information S1, S2, S3). HUC-MSCs were isolated as previously described [14]. Segments of the tissue was cut into pieces (∼1 inch long) and dissected to open the cord vessel. The pieces were placed in 250-mm plastic Petri dishes containing DMEM medium and incubated for approximately 1 day in a 5% CO2 incubator at 37°C. The Wharton’s jelly absorbed DMEM (containing phenol red) and was dissected with a razor; pieces of the outer envelope membrane were cultured after rinsing. Mesenchymal stem cell Expansion Medium (R&D Systems, Inc., Minneapolis, MN, USA) was used for the expansion of hUC-MSCs.Isolated hUC-MSCs displayed a typical fibroblast-like appearance as MSCs. Flow cytometry analysis confirmed the MSC signature of hUC-MSCs at P3 with a high expression of CD105. The contamination of the culture with hematopoietic cells and endothelial cells was excluded by the absence of CD34 and CD31. Moreover, hUC-MSCs can differentiate into osteoblast-like cells, thus demonstrating their multipotent differentiated potential as MSCs (data not shown).The hepatic differentiation efficiency of hUC-MSCs was first evaluated by analyzing hepatocyte-specific gene expression at the mRNA and protein levels. QRT-PCR results demonstrated that the expression of HNF4α, ALB and CK18 was increased 10-fold, 4-fold and 3-fold, respectively, after 1 week of induction and reached the highest level after 26 days (Figure 1A). However, after 26 days of induction, apoptotic hepatic-differentiated hUC-MSCs appeared. The expression of hepatocyte-specific genes in the hepatic differentiated hUC-MSCs was also confirmed by immunofluorescence (Figure 1B).Hepatic differentiation efficiency was also evaluated at the functional level. PAS staining demonstrated that hUC-MSCs submitted to the hepatic differentiation protocol were able to specifically store glycogen compared with undifferentiated hUC-MSCs after induction for 2 weeks (Figure 2A). The ability of hepatic differentiated hUC-MSCs to produce urea was evaluated by exposing the cells to 10 mmol/l ammonium chloride for 24 hours. The urea production ability of hUC-MSCs increased significantly after induction by hepatic differentiation medium and reached the highest level at day 22. At day 26, the urea production ability of the differentiated hUC-MSCs decreased compared to day 22 (Figure 2C). Moreover, after induction for 2 weeks, all hepatic differentiated hUC-MSCs could uptake LDL (Figure 2B). These results indicated that hUC-MSCs differentiated into cells with significant hepatic gene expression and hepatic functions.Informed Consent for Umbilical Cord Donation – Chinese.(TIF)We thank Yongzhan Nie and Zheng Chen for technical guidance during the experiments.